Recent advances in immunotherapy have completely transformed the treatment and prognosis of veterans with advanced melanoma. Checkpoint inhibition immunotherapy using anti-CTLA-4 and anti-PD-1 antibodies has introduced the possibility of cure for a disease that was once practically untreatable. Sadly, the majority of veterans with metastatic melanoma will still succumb despite treatment, and incremental improvements in efficacy have brought increasing risks of autoimmune complications. For veterans reaching the therapeutic limits of checkpoint inhibition, it is clear that a new generation of immunotherapy is needed. In this proposal, we examine an entirely different approach to melanoma immunotherapy. Our vision for this approach fuses a number of long-standing and recent observations in cancer immunology. It is well known that conventional oncological therapy and modern immunotherapy both heighten the immune response to cancer; conventional treatments like radiation or ablation magnify tumor antigen presentation by destroying tumor cells, and immunotherapy potentiates the activation of tumor-reactive T cells. The presentation of tumor antigens and the activation of reactive T cells occur, in large part, in the tumor-draining lymph node ? the front line of contact between cancer and the immune system. In our previous Merit Review, we learned that a combinatorial approach to immunotherapy using checkpoint inhibition with adoptive cell transfer generated a qualitatively stronger immunity to melanoma antigen. We also characterized the unique oncological advantages of tumor-reactive memory T cells over traditional effector T cells for adoptive immunotherapy. In recent studies, we have learned that the combination of effector and memory T cells is strikingly more effective than effector or memory T cells alone. Despite their enormous theoretical promise for immunotherapy, the practical utility of memory T cells is handicapped by the fact that they exist in vanishingly small quantities. Fortunately, some very recent observations have begun to shed light on the cellular metabolic pathways that determine whether activated T cells differentiate into effector or memory T cells; intentional redirection of these pathways can drive T cells toward either phenotype. Interestingly, the metabolic conditions that favor memory T cell development are inherently unfavorable for cancer cell survival. Just as importantly, we have discovered a technique for isolating and expanding large quantities of tumor-reactive effector or memory T cells using cytokine stimulation of lymphocytes harvested from tumor-draining lymph nodes. We envision a new paradigm of melanoma immunotherapy. In this approach, local and systemic oncoimmunological therapies like radiation or tumoral ablation are used with checkpoint inhibition immunotherapy ? not simply to treat tumors, but to also magnify the activation of tumor-reactive T cells within tumor-draining lymph nodes. Next, local injection of nanoparticles carrying targeted modulators of T cell metabolism are taken up into tumors and peritumoral lymphatics ? exerting anti-cancer effects, while driving activated T cells in tumor-draining lymph nodes toward memory phenotype. Patients undergo a minor surgical procedure to excise these tumor-draining lymph nodes, and their lymphocytes are subjected to stimulation and cytokine stimulation protocols with targeted modulators of T cell metabolism to generate massive and parallel populations of effector and memory melanoma-specific T cells. These cells are adoptively transferred back into the patient, taking advantage of the cooperative benefits between checkpoint inhibition and adoptive immunotherapy. Our paradigm focuses on the tumor-draining lymph node, using oncoimmunological therapies to re-engineer this leading edge between cancer and the immune system into a natural generator of optimally therapeutic and personalized T cells. In this proposal, we outline a series of murine and human experiments that will establish the preclinical validity of this next generation of melanoma immunotherapy.